壳聚糖修饰对细胞摄入和细胞毒性的影响

Cellular Uptake and Cytotoxicity of Modified Chitosans as Gene Carriers

目的 研究精氨酸或十六烷基修饰壳聚糖对其细胞摄入和细胞毒性的影响及作用机制。方法 用α-^32P-dATP标记质粒DNA，分别与十六烷基化壳聚糖和精氨酸修饰的壳聚糖通过复凝聚方法形成壳聚糖DNA纳米复合物。采用血管平滑肌A10细胞进行摄入测试，并用β液闪计数仪检测结果。用MTT方法对壳聚糖DNA纳米复合物的细胞毒性进行评价。结果 经^32P标记的DNA与不同修饰的壳聚糖复合所形成的纳米复合物粒径约为55．9—174．9nm，zeta电位约为1．8—10．8mV。细胞摄入实验显示通过精氨酸或十六烷基修饰的壳聚糖与DNA形成的纳米复合物更易于进入细胞。其中十六烷基化壳聚糖DNA纳米复合物（HCNC）在N／P为1：1时细胞摄入量最高，精氨酸修饰的壳聚糖DNA纳米复合物（ACNC）细胞摄人次之，与未经修饰的壳聚糖DNA纳米复合物（UCNC）相比，差异具有显著性（HCNC、ACNC比UCNC高1．3倍，P〈0．05）。细胞毒性实验显示，修饰后的壳聚糖纳米复合物的毒性远远小于商品化的细胞转染试剂Lipofectamine2000。结论 两种修饰对壳聚糖DNA纳米复合物的血管平滑肌细胞摄入有明显促进作用，其作用机制各不相同；同时其细胞毒性远小于商品化的细胞转染试剂Lipofeetamine2000。

Objective To evaluate the effects of arginine modified chitosan or hexadecylated modified chitosan as gene carriers on the cellular uptake by vascular smooth muscle cells and its in vitro cytotoxicity. Methods Plasmid DNA was labeled with α-^32p-dATP and complexed with the modified chitosans or unmodified chitosan to form nanoparticle complexes by complex coacervation method. Uptake of all kinds of chitosan/ DNA nanoparticle complexes （CNC） by A10 cells was measured by β-liquid scintillation counting. The in vitro cytotoxicity of the CNC was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide （MTY） assay. Results The diameters of the CNC ranged from 55.9-174.9 nm and the zeta potentials were from 10. 8 mV for the arginine modified chitosan/DNA nanoparticle complexes （ ACNC ） to 1.8 mV for the hexadecylated chitosan/DNA nanoparticle complexes （HCNC）. The cellular uptake of the modified chitosan/ DNA nanoparticle complexes （MCNC） by A10 cells increased significantly when compared with the unmodified chitosan/DNA nanoparticle complexes （UCNC） （ P 〈 0.05 ）, with the HCNC at N/P ratio of 1 ： 1 and the ACNC at ratio of 8：1 showing the highest cellular uptake （ 1.3 fold higher than UCNC, P 〈 0.05 ）. MCNC were much less cytotoxic when compared with Lipofectamine 2000-DNA nanoparticles. Conclusion DNA nanoparticle complexes prepared with either arginine or hexadecylated modified chitosan can improve the cellular uptake of the DNA, while the in vitro cytotoxicity of both of the modified chitosan is much less than that of Lipofectamine 2000.